Journal of General Virology (2007), 88, 1866–1875
DOI 10.1099/vir.0.82868-0
Susceptibility of cancer cells to herpes simplex
virus-dependent apoptosis
Marie L. Nguyen, Rachel M. Kraft and John A. Blaho
Correspondence
John A. Blaho
Department of Microbiology, One Gustave L. Levy Place, Mount Sinai School of Medicine, New York,
NY 10029-6574, USA
[email protected]
Received 22 January 2007
Accepted 1 March 2007
Apoptosis has recently been associated with herpes simplex virus 1 (HSV-1) latency and disease
severity. There is an intricate balance between pro- and anti-apoptotic processes during HSV-1
infection. When anti-apoptotic pathways are suppressed, this balance is upset and the cells die
by apoptosis, referred to here as HSV-1-dependent apoptosis (HDAP). It has been observed
previously that HeLa cancer cells exhibit an enhanced sensitivity to HDAP. Here, a series of
specific patient-derived cancer cells was utilized to investigate the cell-type specificity of HDAP.
The results showed that a human mammary tumour cell line was sensitive to HDAP, whilst
syngeneic normal cells were resistant. Furthermore, low-passage-number primary human
mammary epithelial cells were resistant to HDAP. When the susceptibility of human colon, brain,
breast and cervical cancer cells was assessed, the only cells insensitive to HDAP were those
resistant to all environmental stimuli tested. This implies that the HDAP resistance was probably
due to mutations in the cellular apoptotic machinery. Thus, the susceptibility of cancer cells to
HDAP requires that they possess a functional ability to undergo programmed cell death.
INTRODUCTION
Apoptosis is initially triggered early in a herpes simplex
virus 1 (HSV-1) infection through the transcription of a
specific immediate-early viral gene, ICP0 (Sanfilippo &
Blaho, 2006; Sanfilippo et al., 2004). During the later stages
of the virus life cycle, anti-apoptotic proteins are synthesized, which prevent apoptotic cell death from proceeding.
This produces a delicate balance of apoptotic signals during
infection that allows the cells to produce progeny virions
prior to apoptotic cell death (reviewed by Nguyen & Blaho,
2007). However, in situations where the preventors are
not produced efficiently, the balance is upset and the
infected cells die by apoptosis. To date, at least one cellular
(Goodkin et al., 2003; Gregory et al., 2004; Yedowitz &
Blaho, 2005) and seven viral survival factors (Aubert &
Blaho, 1999; Aubert et al., 1999; Gupta et al., 2006; Jerome
et al., 1999, 2001; Leopardi & Roizman, 1996; Leopardi
et al., 1997; Perng et al., 2000) have been identified and
implicated in the apoptotic prevention process. One essential viral preventer of apoptosis is the immediate-early
ICP27 protein. ICP27-null viruses are able to trigger but
not prevent apoptotic cell death, and cells infected with
these viruses die (Aubert & Blaho, 1999; Aubert et al.,
2001) through a process that we refer to here as HSV-1dependent apoptosis (HDAP).
Much of the initial characterization of HDAP utilized the
HEp-2 strain of HeLa cells (Chen, 1988; Nelson-Rees et al.,
1974; Ogura et al., 1993) and these studies provided
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information crucial for understanding the viral factors
involved in modulating the process (reviewed by Aubert &
Blaho, 2001). When studies were expanded to include
other cell types, it became apparent that a range of sensitivities to HDAP exists. For instance, Vero cells, which are
a primate kidney cell line typically used for propagating
HSV-1, did not exhibit HDAP at early time points (Aubert
& Blaho, 1999). Further studies revealed that Vero cells do
undergo HDAP, albeit at later times post-infection than
HeLa cells (Nguyen et al., 2005). The inhibition of protein
synthesis during infection reduced HDAP in Vero cells,
demonstrating that proteins newly synthesized early in
infection facilitate HDAP. Synthesis of these proteins is
essential for efficient HDAP in Vero but not HeLa cells,
highlighting a fundamental difference in the way these
two cell lines respond to this process. Although both HeLa
and Vero cells have an indefinite life span (i.e. they are
immortalized), only HeLa cells display the anchorageindependent growth needed to form a tumour (i.e. they are
transformed) (Contreras et al., 1985). In contrast to both
HEp-2 and Vero cells, primary murine and human fibroblast cells were completely resistant to apoptosis induced
by HSV-1 (Aubert & Blaho, 2003).
In this study, we set out to address whether transformation
status could explain the differences in the response to
HDAP. The susceptibility of human cancer cells derived
from various types of tumour was assessed. Cells derived
from normal tissue, peripheral to a mammary tumour,
were resistant to HDAP, whilst the syngeneic cancer cells
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Cell-type differences in HSV-dependent apoptosis
were susceptible, indicating that genetic lesions occurring
during tumorigenesis sensitized these cells. The susceptibility of cells derived from colon, brain, breast and cervical
cancers to HDAP was determined. Two cell lines were
resistant to HDAP, but they were also highly resistant to
exogenous apoptotic stimuli. These resistant cells have
probably acquired additional mutations that target their
cellular apoptotic machinery. Together, these results indicate that the efficiency of the cellular apoptotic response is
a determinant that is capable of altering susceptibility to
HDAP.
METHODS
Cells and viruses. All cells were obtained from ATCC. U373, SK-NSH, RKO and RKO-E6 cells were maintained in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10 % fetal bovine
serum (FBS). Hs578T cells were grown in DMEM containing 10 %
FBS and 0.01 mg bovine insulin ml21. HT-29 and PC-3 cells were
grown in 10 % FBS-containing McCoys 5a or F12K medium, respectively. Hs578Bst cells were grown in Hybri-Care (ATCC) medium
supplemented with 10 % FBS and 30 ng epidermal growth factor
ml21. Hs578Bst cells are a diploid cell line that we obtained from
ATCC at passage 9. In preliminary experiments, we observed that
these cells enter a non-growing state around passage 16, similar to
that observed for primary epithelial cells. Therefore, all experiments
were performed using Hs578Bst cells used at passages prior to passage
14. Human mammary epithelial cells (HMECs) were grown in mammary epithelial growth medium (both from Cambrex). The HMECs
used in this study had undergone approximately four population
doublings since purchase. Vero 2.2 and HEp-2 cells were maintained
in DMEM with 5 % FBS. Vero 2.2 cells (a gift from Saul Silverstein,
Columbia University, NY, USA) are derivatives of Vero cells expressing ICP27 from its viral promoter (Sekulovich et al., 1988). HSV-1
KOS1.1 was the strain of wild-type HSV-1 used in this study. HSV-1
strain vBSD27 is an ICP27-null virus derived from HSV-1 KOS 1.1
containing a replacement of the a27 gene with the Escherichia coli lacZ
gene (Soliman et al., 1997). This virus was propagated and titrated on
Vero 2.2 cells and used to infect cells at an m.o.i. of 10, as reported
previously (Nguyen et al., 2005). CgalD3 was derived from HSV-1
strain 17syn+ and is an IE3 (ICP4)-null virus that has a deletion of
3.6 kb of the coding region of IE3 (Paterson et al., 1990) due to
insertion of the Escherichia coli lacZ gene in the BamHI Z fragment
(Johnson et al., 1992). As described previously (Nguyen et al., 2005),
CgalD3 was propagated and titrated on FO6 cells, which are derived
from Vero cells and express ICP27, ICP4 and ICP0 from their own
promoters (Samaniego et al., 1997). In experiments designed to inhibit protein synthesis, cycloheximide (CHX) was added directly to the
medium at a concentration of 10 mg ml21 1 h prior to infection and
maintained at that level until the time of harvest. As we observed
previously (Aubert & Blaho, 1999), it was expected that certain cells,
particularly highly transformed lines, may have increased resistance to
CHX. With this caveat, we chose the concentration of CHX that is our
standard amount required for complete inhibition of transformed
human HEp-2 cells. Tumour necrosis factor (TNF; 10 ng ml21) plus
CHX (10 mg ml21) or staurosporine (STS; 1 mM) were added to cells
as a positive control for apoptosis induction. Unless otherwise noted,
all cell-culture reagents were obtained from Life Technologies and all
biochemicals from Sigma.
Microscopic analysis and monitoring of chromatin condensation. The morphology of infected cells was documented by phase-
contrast and fluorescence microscopy using an Olympus IX70/
IX-FLA inverted fluorescence microscope. Images were acquired
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using a Sony DKC-5000 digital photo camera linked to a PowerMac
workstation and processed through Adobe Photoshop. For visualization of chromatin condensation in live cells, 5 mg Hoechst 33258
(Sigma) ml21 was added to the medium and allowed to incubate at
37 uC for 30 min. The percentage of nuclei containing condensed
chromatin was determined by dividing the number of brightly
stained, small (condensed) nuclei by the total number of nuclei
(uncondensed plus condensed) in a particular (640) microscopic
field. At least 100 nuclei were counted for each data point. For Fig. 1,
the percentage of chromatin condensation is represented as the
mean±SD of three independent experiments.
Immunoblotting. Whole-cell protein extract was prepared using lysis
buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 % Triton X-100,
1 % deoxycholate, 0.1 % SDS) supplemented with 2 mM PMSF
(freshly prepared stock), 1 % Translysol, 0.1 mM L-1-chloro-3-(4tosylamido)-4-phenyl-2-butanone, 0.01 mM L-1-chlor-3-(4-tosylamido)-7-amino-2-heptanon-hydrochloride, as previously reported
(Nguyen et al., 2005). Protein concentrations were determined using
a modified Bradford protein assay (Bio-Rad Laboratories). Total
protein (20 or 50 mg) was separated on 15 % N,N9-diallyltartardiamide-acrylamide gels and electrically transferred to nitrocellulose.
Pre-stained molecular mass markers were loaded (not shown) and
immunostaining of the actin loading control was carried out.
Membranes were incubated for 1 h at room temperature in blocking
buffer (PBS containing 5 % non-fat, dried milk) and incubated
overnight at 4 uC in primary antibody. Monoclonal antibodies specific
for ICP4, ICP27, gC (all from the Goodwin Institute for Cancer
Research), poly(ADP-ribose) polymerase (PARP) (PharMingen),
procaspase 3 (BD Transduction) and the control actin (Sigma) and
polyclonal antibodies specific for thymidine kinase (TK) and DFF-45
(Santa Cruz) were diluted at a concentration of 1 : 1000 in Trisbuffered saline containing 0.1 % Tween 20 (TBST) and 0.1 % BSA.
After washing in TBST, membranes were incubated with either antimouse or anti-rabbit antibodies conjugated to alkaline phosphatase
(Southern Biotech) diluted in blocking buffer (1 : 1000) for 1 h at
room temperature. Following washing in TBST, immunoblots were
developed in buffer containing 5-bromo-4-chloro-3-indolyl phosphate and 4-nitro blue tetrazolium chloride.
Densitometric analysis. To quantitate the percentage of total
infected cell PARP that was cleaved, densitometry of immune-reactive
PARP was performed as described previously (Aubert et al., 1999).
NIH IMAGE version 1.63 was used to measure the integrated density
(ID) of the 116 kDa uncleaved and 85 kDa cleaved PARP bands.
These values were used to calculate the percentage of PARP cleavage
for each lane using the following formula: % cleavage5[(cleaved
PARP ID)/(cleaved PARP ID plus uncleaved PARP ID)]6100 %.
RESULTS
An intricate balance between pro- and anti-apoptotic
processes exists in HSV-1-infected cells, which enables
productive virus propagation. When anti-apoptotic pathways are suppressed during infection, this balance is upset
and the cells die by apoptosis. In previous studies (Aubert
& Blaho, 2003; Nguyen et al., 2005), we observed that HeLa
cancer cells exhibited an enhanced sensitivity to HDAP,
which implicated oncogenic pathways in HDAP regulation. The goal of this study was to utilize a series of cancer
cells to determine whether their transformation status
was sufficient to be a determinant of apoptotic killing by
HSV-1.
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M. L. Nguyen, R. M. Kraft and J. A. Blaho
Fig. 1. HSV-1-dependent apoptosis occurs in
mammary tumour cells, but not in syngeneic
normal breast cells. (a) Nuclear (Hoechst) and
cellular (phase) morphologies. Hs578T tumour
and Hs578Bst normal cells were visualized at
24 h following infection with wild-type HSV-1
(KOS) or apoptotic vBSD27 (D27) (m.o.i.510)
or at 24 h post-treatment with STS. Magnification, ¾40. Insets inside Hoechst panels
represent enlarged images of nuclei from each
panel to aid in visualization. The values in the
lower right corner of the Hoechst panels
denote the mean±SD of the percentage of
nuclei containing condensed chromatin from
three independent experiments assessed as
described in Methods. (b, c) Immunoblots of
death factors (PARP, DFF-45 and procaspase
3) (b) and viral proteins (ICP4, ICP27, TK and
gC) (c) in Hs578T and Hs578Bst cells at 24 h
post-treatment with STS or post-infection
with HSV-1 (KOS) or vBSD27 (D27) in the
presence (+) or absence (”) of CHX. The
positions of the 116 kDa uncleaved and
85 kDa cleaved PARP products are indicated
in the right-hand margin. Some slight spreading of the bottom of the gel occurred in the
very low molecular mass range such that the
panel showing procaspase 3 does not align
with the other panels.
HSV-1-dependent apoptosis occurs in mammary
tumour cells, but not syngeneic normal breast
cells
Whilst earlier studies provided evidence that the sensitivity
to HDAP is linked to transformation status (Aubert &
Blaho, 2003; Nguyen et al., 2005), a direct comparison of
syngeneic tumour and normal cells was not done. Our first
experiment compared Hs578T mammary tumour cells
with normal epithelial (Hs578Bst) cells derived from tissue
peripheral to the tumour (Hackett et al., 1977). All experiments with the Hs578Bst cells were performed using cells
with passage numbers less than 14. To assess the ability of
these cells to undergo HDAP, Hs578T and Hs578Bst cells
were mock infected or infected with wild-type HSV-1
strain KOS1.1 (KOS) or with an ICP27-null recombinant
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virus, vBSD27. In addition, as we have recently determined
that at least one cell line requires de novo protein synthesis
to undergo HDAP (Nguyen et al., 2005), we also assessed
the role of protein synthesis by performing the experiments
in the presence and absence of the protein synthesis
inhibitor CHX. STS treatment was also used as a positive
control for apoptosis induction. Apoptosis was evaluated at
24 h post-treatment by monitoring morphological changes,
chromatin condensation, procaspase 3 and DFF-45 protein
levels and cleavage of the caspase 3 substrate, PARP, from
its 116 kDa form into an 85 kDa fragment.
Mock-infected Hs578T and Hs578Bst cells were flat and
well spread out and their nuclei exhibited homogeneous
Hoechst staining (Fig. 1a). In contrast, KOS-infected
Hs578T cells exhibited an enlarged, rounded morphology,
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Journal of General Virology 88
Cell-type differences in HSV-dependent apoptosis
and bright Hoechst staining was evident in the periphery of
their nuclei. These morphological changes are characteristic of the cytopathic effect (CPE) that accompanies productive HSV-1 replication (Avitabile et al., 1995; Hampar
& Elison, 1961; Heeg et al., 1986; Roizman, 1962, Roizman
& Roanne, 1964). Although the CPE in Hs578Bst cells was
more subtle than that of KOS-infected Hs578T cells, their
morphology differed from that of mock-infected cells.
Specifically, the nuclei were larger, with brighter Hoechst
staining around the periphery compared with mockinfected cells, which had uniform staining. In addition, a
prominent ridge was evident around the nuclei in the light
microscopy images of KOS-infected Hs578Bst cells, which
is a common feature of cells undergoing productive HSV-1
infection. The KOS-infected cells of both cell types produced similar levels of representative immediate-early
(ICP4 and ICP27), early (TK) and late (gC) viral proteins
(Fig. 1c, lanes 3 and 9). These results indicated that the
Hs578T and Hs578Bst cells were capable of supporting
HSV-1 infection with similar efficiencies. STS-treated
Hs578T and Hs578Bst cells were smaller and irregular
shaped compared with the mock-treated cells (Fig. 1a). In
addition, they exhibited membrane protrusions characteristic of membrane blebbing. The nuclei of STS-treated cells
were smaller in size than those of mock-treated cells and
contained regions of intense Hoechst staining indicative
of chromatin condensation. When this phenotype was
quantified for three independent experiments, the Hs578T
and Hs578Bst cells exhibited 86±14 % and 74±44 %
chromatin condensation, respectively. The lysates of STStreated Hs578T and Hs578Bst cells also displayed a band
corresponding to the cleaved 85 kDa product of PARP, and
procaspase 3 and DFF-45 protein levels were drastically
reduced from that of mock-infected cells (Fig. 1b, compare
lane 7 with 1 and lane 14 with 8). As the STS-treated
Hs578T and Hs578Bst cells exhibited the morphological
and biochemical characteristics of apoptosis, we concluded
that both cell types were capable of undergoing apoptosis.
Thus, the primary Hs578Bst cells were not senescent and
not generally resistant to apoptosis.
Fifty-three per cent of the Hs578T cells infected with
vBSD27 exhibited membrane blebbing and chromatin condensation (Fig. 1a). Additionally, they displayed PARP
cleavage and had lower levels of DFF-45 and procaspase 3
(Fig. 1b, lane 5) than mock-infected cells (Fig. 1b, lane 1).
These results demonstrated that the Hs578T cells were
sensitive to HDAP. Similarly, infection with KOS or
vBSD27 in the presence of CHX led to apoptotic morphologies (data not shown) and reductions in DFF-45 and
procaspase 3 (Fig. 1b, lanes 4 and 6) in these cells.
Although CHX treatment led to some background PARP
cleavage in mock-infected Hs578T cells, significantly more
PARP cleavage was evident in cells treated with KOS plus
CHX and vBSD27 plus CHX (Fig. 1b, compare lane 2 with
lanes 4 and 6). Importantly, significant PARP and complete
DFF and procaspase 3 processing was observed with
vBSD27-infected cells in the absence of CHX, indicating
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that this was not simply due to CHX. Together, these
results demonstrated that Hs578T cells underwent HDAP
in a manner similar to the HEp-2 cells and that they did
not require de novo protein synthesis for this process to
occur.
In contrast, only a very small percentage (4±4 %) of the
vBSD27-infected Hs578Bst cells exhibited chromatin condensation (Fig. 1a). This level was comparable to that seen
in KOS-infected Hs578Bst cells (5±9 %). Furthermore,
neither detectable PARP cleavage nor reductions in DFF-45
or procaspase 3 were observed in Hs578Bst cells that were
infected with vBSD27 (Fig. 1b, compare lanes 12 and 8).
This result indicated that, although the Hs578Bst cells were
sensitive to STS-induced apoptosis, they were resistant to
HDAP. Hs578Bst cells treated with KOS or vBSD27 plus
CHX also failed to undergo apoptosis (Fig. 1b, compare
lanes 11 and 13 with lane 8). Together, the data presented
in Fig. 1 demonstrated that the normal tissue-derived
Hs578Bst cells were resistant to HDAP, whilst the tumourderived Hs578T cells were sensitive.
Primary HMECs are resistant to HDAP
The differential sensitivity of primary Hs578Bst and transformed Hs578T cells suggested that genetic changes occurring during tumorigenesis sensitized the tumour cells to
HDAP. This could reflect a general sensitivity of tumour
cells to pro-apoptotic stimuli, which is the basis of certain
chemotherapy treatments. However, the primary Hs578Bst
cells were originally derived from normal tissue and the
aliquot that we received from ATCC was from passage
number 9. Therefore, it was possible that these cells had
acquired genetic mutations during their subculturing that
had rendered them resistant to HDAP. Thus, we tested the
susceptibility of low-passage-number primary normal
HMECs to HDAP. To accomplish this, primary HMECs
(Cambrex) grown in defined growth medium were infected
with wild-type KOS and vBSD27 at an m.o.i. of 10. Twentyfour hours later, the cells were assessed for chromatin
condensation, PARP cleavage and the presence of viral
proteins. Cells infected with KOS exhibited CPE (Fig. 2a)
and expressed the ICP4, gC and ICP27 viral proteins
(Fig. 2b, lane 2). As expected, ICP4, but not ICP27 or gC,
was detected in the lysate of vBSD27-infected cells (Fig. 2b,
lane 3). These results confirmed that the primary HMECs
were infected efficiently with KOS and vBSD27. However,
the vBSD27-infected primary HMECs did not display
chromatin condensation (Fig. 2a). Furthermore, we did not
detect any PARP cleavage or reductions in DFF-45 and
procaspase 3 protein levels with vBSD27 (Fig. 2b, lane 3).
We observed a similar apoptotic resistance of separate
isolations of primary HMECs (data not shown). We consistently observed increases in the amounts of procaspase 3
and DFF relative to mock- and KOS-infected cells during
vBSD27 infection (Fig. 2b, lane 3). The basis of this is
unknown, but it further confirmed the lack of apoptosis in
these cells. From these findings, we concluded that primary
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M. L. Nguyen, R. M. Kraft and J. A. Blaho
are equally susceptible to HDAP. To gain further insight
into the cancer-cell determinants for susceptibility to
HDAP, we analysed a broader range of tumour cells.
Specifically, we assessed the sensitivity of cell lines derived
from colon (HT-29, RKO and RKO-E6), prostate (PC-3)
and brain (SK-N-SH and U373) tumours to HDAP. HEp-2
cells were used as a control cell line that was known to be
sensitive to HDAP (Aubert & Blaho, 2003).
Each cell line was infected with KOS, vBSD27 and/or
another recombinant virus that lacked expression of ICP4,
CgalD3. Like vBSD27, CgalD3 triggers but does not prevent
apoptosis during infection (Aubert & Blaho, 2003). We
also assessed the role of protein synthesis during HDAP
treatment in these cell lines by adding CHX to a subset of
the infections. STS and/or TNF plus CHX were used as
positive controls for apoptosis. At 24 h p.i., chromatin
condensation was monitored via Hoechst staining. Subsequently, cells were harvested and immunoblotted for the
accumulation of viral proteins and biochemical markers of
apoptosis. Cell morphologies and immunoblot results from
the HT-29, RKO and SK-N-SH cells are presented in Figs 3
and 4. The results from PC-3 and U373 cells are displayed
in Fig. 5. The HEp-2 cells are presented in each figure for
comparison.
Fig. 2. Primary HMECs are resistant to HSV-1-dependent apoptosis. (a) Nuclear (Hoechst) and cellular (phase) morphologies.
Primary HMECs were visualized at 24 h following HSV-1 (KOS)
and vBSD27 (D27) infection (m.o.i.510). The values in the lowerright corner of the Hoechst panels denote the mean±SD of the
percentage of nuclei containing condensed chromatin, assessed
as described in Methods. Magnification, ¾40. (b) Immunoblots of
death factors (PARP, procaspase 3, DFF-45), viral proteins (ICP4,
ICP27, gC) and the control actin at 24 h post-infection. The
positions of the 116 kDa uncleaved and 85 kDa cleaved PARP
products are indicated in the right-hand margin.
HMECs are resistant to HDAP. These results, along with
those in Fig. 1, are significant as they represent the first
characterization of primary human epithelial cells infected
with an HSV strain that results in apoptosis of at least two
(HeLa and Hs578T) types of cancer cell.
The susceptibility of cancer cells to HSV-1dependent apoptosis correlates with sensitivity to
exogenous environmental apoptotic inducers
The results described for Fig. 1 demonstrated that at least
one breast cancer cell line is sensitive to HDAP. Previously
published studies have shown that HeLa/HEp-2 cancer and
143 tumour cell lines, typically used to study HSV-1 replication, are sensitive to HDAP (Aubert & Blaho, 1999, 2003;
Koyama & Adachi, 1997). However, transformed human
embryonic kidney 293 cells and caspase 3-null MCF-7
breast cancer cells are resistant to HDAP (Aubert & Blaho,
2003; Kraft et al., 2006), indicating that not all tumour cells
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All of the cell lines tested displayed CPE (Figs 3 and 5a) and
had similar levels of accumulation of the ICP27 viral protein following KOS infection (Fig. 4 and Fig. 5b–d, lane 3),
indicating that they were equally susceptible to viral infection. STS treatment led to abundant apoptotic morphologies in the HT-29, RKO and SK-N-SH cell lines (Fig. 3).
Additionally, 50 % of RKO and 30 % of HT-29 nuclei exhibited chromatin condensation following STS treatment.
It was not possible to assess chromatin condensation in
SK-N-SH cells in this manner due to a high level of
background Hoechst staining in these live cells (data not
shown). STS induced almost complete (.95 %) PARP
cleavage in HT-29, RKO and SK-N-SH cells, as well as
reductions in procaspase 3 levels (Fig. 4b and d, lane 7;
Fig. 4c, lane 9). Together, these results indicated that these
cell lines were sensitive to STS-induced apoptosis. TNF
plus CHX treatment similarly induced apoptotic morphologies (data not shown) and death factor processing in
these cells (Fig. 4b, lane 8, and data not shown).
Like HEp-2 cells, which are susceptible to HDAP, vBSD27infected HT-29, RKO and SK-N-SH cells exhibited membrane blebbing (Fig. 3). Infection with vBSD27 led to 50
and 23 % of nuclei with condensed chromatin in HT-29
and RKO cells, respectively. The lysates from all of these
cell lines also displayed PARP cleavage levels of between
51 and 60 % and small reductions in procaspase 3 when
infected with vBSD27 (Fig. 4b, c, lane 5). In addition, RKO
cells infected with CgalD3 exhibited 37 % PARP cleavage
(Fig. 4c, lane 7), indicating that these cells are susceptible
to HDAP induced by multiple recombinant viruses. We
observed that infection with KOS led to 94 % PARP
cleavage in the SK-N-SH cells. Together, these results
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Cell-type differences in HSV-dependent apoptosis
Fig. 3. Cancer cells sensitive to exogenous apoptotic stimuli are sensitive to HSV-1-dependent apoptosis. Cellular (phase)
and nuclear (Hoechst) morphologies of HEp-2, HT-29, SK-N-SH and RKO cells at 24 h following infection with wild-type HSV1 (KOS) or vBSD27 (D27) at an m.o.i. of 10 or treatment with STS. Magnification, ¾40. The values in the lower right-hand
corner of the Hoechst panels denote the percentage of nuclei containing condensed chromatin assessed as described in
Methods.
Fig. 4. Cancer cells sensitive to exogenous apoptotic stimuli are sensitive to HSV-1-dependent apoptosis. Immunoblots of
death factors (PARP and procaspase 3) and viral proteins (gC, ICP27, ICP4 and VP22) from HEp-2 (a), HT-29 (b) RKO (c) and
SK-N-SH (d) cells at 24 h post-treatment with STS or TNF or infection with HSV-1 (KOS), vBSD27 (D27) or CgalD3 (D3) at an
m.o.i. of 10 in the presence (+) or absence (”) of CHX. The positions of the 116 kDa uncleaved and 85 kDa cleaved PARP
products are indicated in the right-hand margin. The percentage of PARP cleavage (%Cleav) was calculated as described in
Methods.
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M. L. Nguyen, R. M. Kraft and J. A. Blaho
demonstrated that HT-29, RKO and SK-N-SH cell lines are
sensitive to HDAP. Other investigators using different
assays have observed low levels of apoptosis in wild-type
HSV-infected SK-N-SH cells (Galvan & Roizman, 1998;
Peng et al., 2005). It should be noted that our SK-N-SH
cells were used directly from ATCC and were at low passage
(,20). Therefore, our results seemed to indicate that the
SK-N-SH cells are unable to set up a perfect apoptotic
balance, even in the presence of viral apoptotic preventors.
This may be due to a heightened sensitivity for apoptosis in
these cells, as even mock-infection led to a relatively high
level of PARP cleavage (29 %, Fig. 4d, lane 1).
RKO, HT-29 and SK-N-SH cells also demonstrated apoptosis when infected with vBSD27 or KOS in the presence of
CHX (data not shown and Fig. 4b–d, lanes 4 and 6),
consistent with HDAP occurring independently of de novo
protein synthesis in these cells. RKO cells expressing the
human papillomavirus E6 protein (RKO-E6) exhibited an
identical response to RKO cells with respect to both STS
and HDAP (data not shown). From these results, we concluded that certain colon and brain tumour-derived cells
can respond to HDAP.
In contrast, PC-3 and U373 cells did not exhibit substantial
apoptotic morphology (Fig. 5a), PARP cleavage, or a
reduction in procaspase 3 when treated with STS (Fig. 5c,
d, lane 7) or TNF plus CHX (Fig. 5d, lane 8, and data not
shown), indicating that these cell lines are more resistant
than the aforementioned cell lines. Strikingly, the PC-3 and
U373 cells displayed little to no membrane blebbing following infection with vBSD27. Only 5 % of the vBSD27infected PC-3 cells displayed chromatin condensation
(Fig. 5a). Furthermore, PARP was found only in the
Fig. 5. Cancer cells that are resistant to
exogenous apoptotic stimuli are resistant to
HSV-1-dependent apoptosis. (a) Cellular
morphologies of HEp-2, PC-3 and U373 cells
were visualized at 24 h following HSV-1
(KOS) and vBSD27 (D27) infection (m.o.i.5
10) or 24 h post-treatment with STS. Magnification, ¾40. The values in the lower-right
corner of the Hoechst panels denote the
percentage of nuclei containing condensed
chromatin assessed as described in Methods.
(b)–(d) Immunoblots of death factors (PARP
and procaspase 3) and viral proteins (ICP4,
ICP27 and gC) from HEp-2 (b), PC-3 (c) and
U373 (d) cells at 24 h post-treatment with
STS or infection with HSV-1 (KOS) or
vBSD27 (D27) in the presence (+) or
absence (”) of CHX. The positions of the
116 kDa uncleaved and 85 kDa cleaved
PARP products are indicated in the right-hand
margin. The percentage of PARP cleavage
(%Cleav) was calculated as described in
Methods.
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Journal of General Virology 88
Cell-type differences in HSV-dependent apoptosis
uncleaved form and procaspase 3 levels did not change
following vBSD27 infection (Fig. 5c, d). This result indicated that PC-3 and U373 cells are resistant to HDAP. KOS
and vBSD27 infections in the presence of CHX also failed
to cause apoptosis in these cells. Together, the results from
Figs 3–5 demonstrated a correlation between the sensitivity
to HDAP and the response to environmental apoptotic
stimuli in cancer cells.
DISCUSSION
HSV modulates apoptosis during the course of its productive infection (reviewed by Nguyen & Blaho, 2007) and
this probably plays an important role in the pathogenesis of
herpesviral disease (Miles et al., 2003; Sabri et al., 2006).
However, recent studies have suggested that there is an
unexpected pattern of cell susceptibility to HDAP, as
tumour cells appear to be exquisitely sensitive to this celldeath process (Aubert & Blaho, 2003). Accordingly, the
goal of this study was to assess whether cell transformation
status was the sole determinant of HDAP. Our key findings
may be summarized as follows.
Sensitivity to environmentally induced apoptosis predicts
cancer cell susceptibility to HDAP. We now have a large
body of information on the response of numerous cell
types to HDAP (Table 1). The striking finding is that cells
that are sensitive to apoptotic cell death triggered by
exogenous agents are also able to be killed by HDAP. All of
these HDAP-susceptible cells were treated with and found
to be sensitive to the intrinsic inducer STS. Of these cells
that were also treated with TNF plus CHX, this group was
also sensitive to this extrinsic method of induction. Thus,
these cells possess the necessary internal apoptotic machinery to respond to all types of pro-apoptotic stimuli. It has
recently been shown that HDAP occurs as a result of cytochrome c release from mitochondria, which occurs independently of caspase activation, and, thus, implicates the
intrinsic apoptotic pathway as the response to virus (Aubert
et al., 2007). Due to the implicit cross-talk that occurs from
the extrinsic to the intrinsic pathways (reviewed by
Sanfilippo & Blaho, 2003), we must conclude that, in order
for a cancer cell to be susceptible to HDAP, it must possess
the intact cellular machinery of the mitochondrial-dependent apoptotic cascade. Our findings should be of interest to
those studying virus-induced apoptosis and the virotherapy
of cancer. We have already shown that viruses singly deleted
for either the HSV ICP4 (this study, and Aubert & Blaho,
2003; Nguyen et al., 2005) or ICP22 (Aubert et al., 1999;
Sanfilippo & Blaho, 2006) regulatory protein also possess the
ability to induce HDAP in certain human tumour cells. It is
conceivable that other viruses possessing deletions in certain
accessory apoptosis prevention factors (reviewed by Aubert
& Blaho, 2001; Goodkin et al., 2004), such as US3 (Jerome
et al., 1999; Leopardi et al., 1997), might have some level of
HDAP efficacy.
Table 1. Cancer cell susceptibilities to HDAP correlate with sensitivities to environmentally induced apoptosis
Cell name
Origin
HeLa/HEp-2
HeLa/S3
HT-29
RKO
RKO-E6
Hs578T
MCF-7
MCF-7/C3
SK-N-SH
Jurkat
U373
PC3
HEK293
HEK293T
HMEC
Hs578Bst
HFF
MEF
Vero
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Mouse
Monkey
Type*
Cervix cancer
Cervix cancer
Colon cancer
Colon cancer
Colon cancer
Breast cancer
Breast cancer
Breast cancer
Brain cancer
T-cell tumour
Brain cancer
Prostate cancer
Kidney tumour
Kidney tumour
Primary epithelial
Primary epithelial
Primary fibroblast
Primary fibroblast
Immortalized fibroblast
HDAPD
Environmentald
Reference
+
+
+
+
+
+
2
+
+
+
2
2
2
2
2
2
2
2
+
+
+
+
+
+
+
2
+
+
+
2
2
2
2
+
+
+
+
+
Aubert & Blaho (1999); Koyama & Adachi (1997)
Aubert & Blaho (1999)
This study
This study
This study
This study
Kraft et al. (2006)
Kraft et al. (2006)
Galvan & Roizman (1998) and this study
Jerome et al. (1999)
This study
This study
Aubert & Blaho (2003)
Aubert & Blaho (2003)
This study
This study
Aubert & Blaho (2003)
Aubert & Blaho (2003)
Aubert & Blaho (1999); Leopardi & Roizman
(1996); Nguyen et al. (2005)
*Cells were patient-derived cancer, laboratory-transformed or -immortalized, and primary.
DSusceptibility to HDAP was determined following infection with various apoptotic HSV strains.
dSensitivity to environmental stimulators of apoptosis was assayed using various methodologies and compared with HDAP as described in the
appropriate citations.
http://vir.sgmjournals.org
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1873
M. L. Nguyen, R. M. Kraft and J. A. Blaho
Not all cancer or tumour cells die by HDAP. In contrast,
those cancer cells that exhibited resistance to HDAP seemed
to possess a general apoptotic defect (Table 1). Of course,
this conclusion carries at least one major caveat. Whilst we
cannot rule out the possibility that PC-3 and U373 cells can
undergo apoptosis when exposed for longer time periods
or with higher doses of apoptotic inducers, this may not be
significant if the time that it takes to detect markers of
apoptosis exceeds the virus replication cycle. Nevertheless,
it was clear that such cells were more resistant to STS and
TNF plus CHX treatments than the cells that were sensitive
to HDAP. Although initial oncogene activation or tumour
suppressor inhibition commonly renders certain cells more
susceptible to apoptosis, it has been postulated that subsequent mutations selected for during tumorigenesis render
these cells less sensitive (reviewed by Brown & Wouters,
1999). Importantly, these mutations often affect the functions of proteins central to apoptosis execution. In fact,
PC-3 cells have been reported to overexpress the antiapoptotic Bcl-2 family member Bcl-XL (Liu & Stein, 1997),
which is probably the reason they failed to display HDAP.
It is conceivable that the U373 cells contain similar genetic
alterations that render them highly resistant to apoptosis.
Further support for this mutational defect model comes
from our recent findings that the caspase 3-null breast
cancer cells MCF-7 are resistant to HDAP (Table 1).
However, MCF-7 cells that were reconstituted for caspase 3
(MCF-7/C3) underwent HDAP (Kraft et al., 2006). In
the case of the laboratory-transformed HEK 293 cells, the
basis of the apoptotic resistance is the integrated (Graham
et al., 1977) presence of adenovirus anti-apoptotic genes
(reviewed by Lichtenstein et al., 2004). In summary, it is
likely that mutations in the cellular apoptotic machinery
are responsible for the cancer cell resistance to HDAP
found in this study.
All primary cells tested were resistant to HDAP. The fact
that a syngeneic pair of tumour and normal cells exhibited
opposite responses to HDAP treatment strongly argues that
alterations in cancer-related genes are responsible for the
tumour-specific cell death. The inability of primary cells to
die by HDAP seemed to be specific to the virus as they were
all still sensitive to other environmental apoptotic inducers
including STS and TNF plus CHX (Table 1). The fact that
these cells were able to die by exogenous apoptosis induction indicated that they were not senescent and did not
possess a general resistance.
The consistently reproducible inability of primary cells to
die by HDAP represents one of the most intriguing and
complicated facets of the analysis of apoptosis during HSV
infection. Recognition of this fact is important in interpreting HSV apoptosis results using MEF cells, especially
those derived from knockout mice. The fact that primary
human fibroblast and epithelial cells respond to HSV in a
manner different from human cancer cells, even though
they all are sensitive to environmental pro-apoptotic stimuli,
emphasizes the importance of cellular pathways targeted in
oncogenesis as central determinants of productive HSV
1874
replication. Future investigations in our group are focusing
on defining the nature of these responses.
Together, the data presented here and in previous publications demonstrate that there are three distinct responses to
HDAP. In general, most patient-derived cancer cells appear
to be exquisitely sensitive to this death stimulus, primary
cells derived from normal tissue are resistant, and immortalized but non-transformed cell lines may display an
intermediate susceptibility. Here, we provide evidence that
disruptions in the cellular apoptotic machinery probably
suppress HDAP in cancer cells. Further elucidation of the
exact mechanisms mediating the cell-type-dependent outcome of HSV infection will require the development of
appropriate biochemical and molecular genetic systems
based on our results.
ACKNOWLEDGEMENTS
We thank Elise Morton (MSSM) for expert technical cell-culture
assistance and Martine Aubert, Ed Goodwin and Dan DiMaio for
critical comments. These studies were supported in part by grants
from the USPHS (A138873 and AI48582 to J. A. B.). M. L. N. was
supported in part by USPHS Institutional Research Training Awards
(AI07647 and CA088796). R. M. K. was supported in part by an
Undergraduate Research Fellowship from the Howard Hughes
Medical Institute to Manhattan College, Riverdale, NY, USA.
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